Medium for the specific detection of resistant microorganisms

ABSTRACT

A method for distinguishing among a first group of microorganisms belonging to a first taxon of Gram negative bacteria, the first group of bacteria exhibiting a mechanism of resistance to a treatment; a second group of microorganisms belonging to a second taxon of Gram negative bacteria, the second taxon of bacteria being different than said first taxon, and exhibiting a mechanism of resistance to a treatment identical to the mechanism of the first group; and a third group of Gram negative bacteria that is not resistant to the treatment.

CROSS-REFERENCE TO PRIOR APPLICATIONS

This is a continuation of application Ser. No. 16/678,502 filed on Nov.8, 2019, which is a continuation of application Ser. No. 15/330,777filed Nov. 7, 2016, which is a continuation of application Ser. No.14/715,222 filed May 18, 2015, which is a continuation of applicationSer. No. 13/539,083 filed Jun. 29, 2012, which is a divisional ofapplication Ser. No. 11/794,907 filed Jul. 9, 2007, which is a NationalStage Application of PCT/FR2006/050109 filed Feb. 9, 2006, and claimsthe benefit of French Application Nos. 05.50394 filed Feb. 10, 2005 and05.53049 filed Oct. 7, 2005. The entire disclosures of the priorapplications are hereby incorporated by reference herein in theirentirety.

The field of the invention is that of microbiological analysis by meansof biochemistry, and in particular the detection and identification ofmicroorganisms, for instance of bacteria or yeasts.

Bacterial resistance to antibiotics is a major public health problem.The resistance of infectious microorganisms to a treatment has developedat the same time as anti-infectious molecules and today represents amajor obstacle in therapeutics. This resistance is responsible for manyproblems, including difficulties in detection in the laboratory, limitedtreatment options and a deleterious impact on clinical outcome. Inparticular, the rapid and irrepressible increase in the resistance ofpathogenic bacteria, over the last 20 years, represents one of the majorcurrent problems in medicine. Infections caused by these organisms areresponsible for extended periods of hospitalization and are associatedwith high morbidity and mortality rates, following therapeutic failures.

Several resistance mechanisms can be involved simultaneously in abacterial strain. They are generally classified in 3 categories:deficient penetration of the antibiotic into the bacterium, inactivationor excretion of the antibiotic by bacterial enzymatic systems, and lackof affinity between the bacterial target and the antibiotic.

Enzymatic inactivation is the most common mechanism of acquiredresistance in terms of number of species and of antibiotics involved.Thus, chromosomal class C cephalosporinases today constitute one of thepredominant resistance mechanisms of gram-negative bacteria, thebacteria expressing such enzymes being resistant to cephalosporins.Similarly, β-lactamases are enzymes expressed by certain bacteria,capable of hydrolyzing the C—N bond of the β-lactame ring, the basicstructure of antibiotics of the β-lactamine family, so as to give amicrobiologically inactive product. Several β-lactamase inhibitors(BLIs), such as clavulanic acid (CA), tazobactam and sulbactam, havebeen developed in order to increase the antimicrobial activity andbroaden the spectrum of the β-lactamines which are associated therewith.They act as a suicide subject for β-lactamases, and prevent enzymaticdegradation of the antibiotics and allow them to become effectiveagainst bacteria that were initially resistant. However, by virtue ofthe persistent exposure of strains to antibiotic pressure, the bacteriaexpress their ability to adapt through the continuous and dynamicproduction of β-lactamases, which evolves at the same time as thedevelopment of new molecules.

Gram-negative bacteria which produce high-level chromosome class Ccephalosporinases (reference is made to HL Case bacteria), and alsogram-negative bacteria which produce extended-spectrum β-lactamase(reference is then made to ESBL bacteria) have, as a result, become anincreasing threat, in particular because the number of bacterial speciesconcerned is increasing. HL Case and ESBL bacteria are resistant totreatments based on 1st- and 2nd-generation penicillins andcephalosporines, but also on 3rd-generation cephalosporines (C3G)(cefotaxim CTX, ceftazidime CAZ, cefpodoxime CPD, ceftriaxone CRO) andmonobactams (aztreonam ATM). On the other hand, 7α-methoxycephalosporins(cephamycins: cefoxitin, cefotetan) and carbapenems (imipenem,meropenem, ertapenem) generally conserve their activity. ESBLs areinhibited by β-lactamase inhibitors (BLIs), which makes it possible todifferentiate them from other cephalosporinases.

These bacteria thus most commonly simultaneously express resistances toseveral treatments, which poses difficulties in setting up a relevanttreatment and avoiding therapeutic failures. An Escherichia colibacterium can thus be HL Case and ESBL. In addition, since ESBL-positiveenterobacteria have a tendency to disseminate the resistance by clonaltransmission of strains or conjugative plasma transfer, they represent aproblem in terms of controlling infections. In most studies, Escherichiacoli and Klebsiella pneumoniae remain the most common ESBL-producingspecies. However, over the last few years, ESBLs have greatly broadenedtheir panel of host species. In fact, many species of enterobacteria andof nonfermenting gram-negative bacilli (such as Pseudomonas aeruginosa)have also been reported to ESBL producers.

In addition to these ESBL bacteria, mention may also be made ofStaphylococcus aureus bacteria, which are also pathogenic bacteria thatdevelop many mechanisms of resistance, such as resistance tomethicillin, penicillin, tetracycline, erythromycin, or vancomycin.Enterococcus faecium is another multiresistant bacterium found in thehospital environment, which can be resistant to penicillin, vancomycinand linezolide. Mycobacterium tuberculosis is commonly resistant toisoniazid and to rifampicin. Other pathogens offer certain resistances,such as Salmonella, Campylobacter and Streptococcus.

It therefore becomes essential, from a public health point of view, tobe able to identify such microorganisms, and such resistance mechanisms,as rapidly as possible.

In general, the search for microorganisms resistant to a treatment iscarried out according to the following steps:

-   -   1. Taking a biological sample that may contain said        microorganisms;    -   2. Seeding and incubating a culture medium (18 to 48 h) in order        to induce exponential growth of the microorganisms;    -   3. Pinpointing, on the culture media, colonies of potentially        significant microorganisms;    -   4. Characterizing the microorganism species;    -   5. Identifying the mechanisms of resistance of the        microorganisms analyzed, their biological significance and,        optionally, the appropriate therapy.

This succession of steps involves a considerable amount of time betweentaking the sample that may contain microorganisms and prescribing atreatment that is appropriate for the patient. Furthermore, the usermust generally perform steps for transferring microorganims from a firstmedium to a second medium manually, which can induce problems, inparticular, of contamination, but also risks to the handler's health.

By way of example, in order to detect the presence of broad-spectrumbeta-lactamases (ESBLs) in strains of Escherichia coli and Klebsiellapneumoniae, use may be made of a diffusion technique as described in thepublication by Jacoby & Han (J Clin Microbiol. 34(4): 908-11, 1996),which does not however give any information regarding the identificationof the strains tested: it is possible to determine whether or not thebacterium is a ESBL-producing bacterium, but it is not possible todistinguish whether such a bacterium is an Escherichia coli or aKlebsiella pneumoniae.

Metabolic substrates are also used for detecting the presence of ESBLsor HL cases. In this respect, AES laboratories proposes a medium in abiplate combining a Drigalski medium with cefotaxim and a MacConkeymedium with ceftazidime. The Drigalski and MacConkey media make itpossible to reveal lactose acidification, a metabolism which is presentin a very large number of enterobacterial species. However, such amedium only makes it possible to distinguish resistant bacteria fromnon-resistant bacteria, and does not make it possible to distinguishbacteria expressing a ESBL from those expressing an HL Case. Neitherdoes this medium make it possible to identify specific bacterialspecies, nor does it make it possible, for example, to discriminatebetween E. coli bacteria and K. pneumoniae bacteria.

In the case of the detection of resistance mechanisms other than ESBL,mention may be made of patent application EP0954560, which relates tothe search for Vancomycin-resistant enterococcal, by combiningVancomycin with a chromogenic media that reveals two enzymaticactivities β-glucosidase and pyrrolidonyl arylamidase). However, thischromogenic medium makes it possible to determine only whether or notthe vancomycin-resistant strains belong to the Enterococcus genus, butdoes not make it possible to identify the species or the resistancemechanisms involved, in particular if it is a question of an acquired orwild-type resistance.

Thus, the characterization of a species of microorganism, and then theidentification of its resistance to a treatment, is long and laborious.If the laboratory gives the clinician a positive screen, whereas theisolate is in fact free of resistant microorganisms, this can lead toneedless and inappropriate treatment. Conversely, not communicating apositive screen, which is subsequently confirmed, delays the setting ofthe isolation of the patient (and possibly an appropriate therapy) byone day. This shows the need for a rapid and reliable confirmation test.

The present invention therefore proposes to improve the prior art byproviding a novel diagnostic tool which allows a gain in time, inreliability and in relevance with respect to the therapy implemented.Our invention makes it possible, in a single step, to identify thespecies of microorganisms present in a sample, and to determine theirmechanism of resistance in order to propose a treatment appropriate toeach patient. This invention is particularly suitable for discriminatingvarious species of microorganisms, which have various mechanisms ofresistance to various treatments, but all of which may be present in thesame sample.

Before going any further in the disclosure of the invention, thefollowing definitions are given in order to facilitate understanding ofthe invention:

The term “culture medium” is intended to mean a medium comprising allthe elements required for the survival and/or the growth ofmicroorganisms. The culture medium according to the invention maycontain any possible additives, for instance: peptones, one or moregrowth factors, carbohydrates, one or more selective agents, buffers,one or more gelling agents, etc. This culture medium may be in liquidform or in gel form which is ready to use, i.e. ready for seeding in atube or flask or on a Petri plate.

For the purpose of the present invention, the term “microorganism”covers gram-positive or gram-negative bacteria, yeasts and, moregenerally, organisms that are generally unicellular, invisible to thenaked eye, which can be multiplied and handled in the laboratory.

By way of gram-negative bacteria, mention may be made of bacteria of thefollowing genres: Pseudomonas, Escherichia, Salmonella, Shigella,Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus,Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria,Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella,Pasteurella, Providencia, and Legionella.

By way of gram-positive bacteria, mention may be made of bacteria of thefollowing genre: Enterococcus, Streptococcus, Staphylococcus, Bacillus,Listeria, Clostridium, Gardnerella, Kocuria, Lactococcus, Leuconostoc,Micrococcus, Mycobacteria and Corynebacteria.

By way of yeasts, mention may be made of yeasts of the following genre:Candida, Cryptococcus, Saccharomyces and Trichosporon.

The term “biological sample” is intended to mean a clinical sample,derived from a specimen of biological fluid, or a food sample, derivedfrom any type of food. This sample may thus be liquid or solid andmention may be made, in the nonlimiting manner, of a clinical blood,plasma, urine or faeces sample, nose, throat, skin, wound orcephalospinal fluid specimens, a food sample from water, from drinkssuch as milk or a fruit juice; from yoghurt, from meat, from eggs, fromvegetables, from mayonnaise, from cheese; from fish, etc., a food samplederived from a feed intended for animals, such as, in particular, asample derived from animal meals.

The term “mechanism of resistance” is intended to mean any type ofdevice which allows a microorganism to render a treatment partially orcompletely ineffective on said microorganism, guaranteeing its survival.The mechanisms of resistance are generally divided up into threecategories: deficient penetration of the antibiotic into the bacterium,inactivation or excretion of the antibiotic by means of bacterialenzymatic systems, and lack of affinity between the bacterial target andthe antibiotic.

By way of indication, mention may in particular be made of mechanisms ofresistance related to the expression of an enzyme belonging to thebroad-spectrum β-lactamase group; of an enzyme belonging to thechromosomal high level class C cephalosporinase group; mechanisms ofresistance to glycopeptides, preferably developed by bacteria belongingto the Enterococcus genus.

Mention will also be made of mechanisms of resistance to methicillin,penicillin, tetracycline, erythromycin, or vancomycin when themicroorganism is a Staphylococcus aureus bacterium.

Mention will also be made of mechanisms of resistance to penicillin,vancomycin and linezolide when the microorganism is an Enterococcusfaecium bacterium.

Mention will also be made of mechanisms of resistance to amphotericin Bor to antifungals of the azole family when the microorganism is a yeast.

Finally, mention will be made of mechanisms of resistance to isoniazidand to rifampicin when the microorganism is a Mycobacterium tuberculosisbacterium.

The term “treatment” is intended to mean a treatment capable ofpreventing or reducing the growth of microorganisms derived from apatient. This treatment may comprise in particular antimicrobialcompounds, such as antibiotics, for instance penicillins, conventionalcephalosporins, broad-spectrum cephalosporins, monobactams,glycopeptides or aminosides, or such as antifungals orresistance-inhibiting compounds. It should be noted that this treatmentcan also comprise the isolation of the patient, thereby preventingpropagation of the microorganism among other patients.

The term “substrate” which allows the detection of an enzymatic ormetabolic activity is intended to mean any molecule capable of directingor indirectly generating a detectable signal due to an enzymatic ormetabolic activity of the microorganism.

When this activity is an enzymatic activity, reference is then made toan enzymatic substrate. The term “enzymatic substrate” is intended tomean any substrate that can be hydrolyzed by an enzyme into a productthat allows the direct or indirect detection of a microorganism. Thissubstrate comprises in particular a first part that is specific for theenzymatic activity to be revealed and a second part that acts as alabel, hereinafter referred to as labeling part. This labeling part maybe chromogenic, fluorogenic, luminescent, etc. As chromogenic substratesuitable for solid supports (filter, agar, electrophoresis gel), mentionmay in particular be made of substrates based on indoxyl and itsderivatives, and substrates based on hydroxyquinoline or escultin andtheir derivatives, which allow the detection of osidase and esteraseactivities. Mention may also be made of substrates based on nitrophenoland nitroaniline and derivatives, making it possible to detect osidaseand esterase activities in the case of substrates based on nitrophenol,and peptidase activities in the case of substrates based onnitroaniline. Finally, mention may be made of substrates based onnaphtol and naphtylamine and their derivatives, which make it possibleto detect osidase and esterase activities via naphtol, and peptidaseactivities via naphtylamine. This substrate may allow, in particular,but in a nonlimiting manner, the detection of an enzymatic activity suchas the activity of an osidase, peptidase, esterase, etc. The enzymaticsubstrate can also be a natural substrate of which the product ofhydrolysis is detected directly or indirectly. As natural substrate,mention may in particular be made of tryptophan for detectingtryptophanase or desaminase activity, a cyclic amino acid (tryptophan,phenylalanine, histidine, tyrosine) for detecting desaminase activity,phosphatidyl inositol for detecting phospholipase activity, etc. Whenthis activity is a metabolic activity, the substrate is then a metabolicsubstrate, such as a source of carbon or of nitrogen, coupled to anindicator that produces a coloration in the presence of one of themetabolic products.

According to a preferred embodiment of the invention, said first and/orsecond enzymatic or metabolic activity is an enzymatic activitypreferably chosen from the enzymatic activities: beta-glucosidase,desaminase, beta-glucuronidase, beta-galactosidase, alpha-glucosidase,alpha-galactosidase, hexosaminidase, N-acetyl-hexosaminidase,phosphatase, esterase, and aminopeptidase.

For example, for detecting E. coli, use is preferably made ofbeta-glucuronidase or β-galactosidase or tryptophanase or desaminaseactivity; for detecting Proteus, use is preferably made of desaminaseactivity; for detecting enterococci, use is preferably made ofbeta-glucosidase activity. For Candida albicans, hexosaminidase ispreferred, for Listeria monocytogenes, phospholipase is preferred, forsalmonellae, esterase is preferred, for Pseudomonas aeruginosa, esteraseor β-alanine aminopeptidase is preferred, for Staphylococcus aureusphosphatase or alpha-glucosidase is preferred.

The expression “marker for differentiating” two groups of microorganismsis intended to mean a compound which does not have the same propertieson a first and on a second group.

This compound may thus be:

-   -   a specific substrate;    -   an inhibitor of a mechanism of resistance, which then makes it        possible to inhibit the growth of the organisms developing a        specific resistance, without any discrimination of the        microorganism species.

In the case of the use of a specific substrate, use is preferably madeof beta-glucuronidase, beta-galactosidase, tryptophanase or desaminaseactivity for detecting E. coli, use is preferably made of desaminaseactivity for detecting Proteus, use is preferably made ofbeta-glucosidase activity for detecting enterococci. For Candidaalbicans, hexosaminidase is preferred, for Listeria monocytogenes,phospholipase is preferred, for salmonellae, esterase is preferred, forPseudomonas aeruginosa, esterase or β-alanine aminopeptidase ispreferred, for Staphylococcus aureus, phosphatase or alpha-glucosidaseis preferred.

In the case of the use of an inhibitor of a mechanism of resistance, useis preferably made of:

-   -   clavulanic acid, tazobactam or sulbactam when the first group        and/or the second group comprises a mechanism of resistance        induced by an expression of β-lactamases. The clavulanic acid        concentration in the medium is then preferably between 0.05 and        32 mg/l, preferably between 0.1 and 8 mg/l, and even more        preferably between 0.25 and 6 mg/l;    -   cloxacillin or dicloxacillin when the first group and/or the        second group comprises a mechanism of resistance induced by the        expression of cephalosporinases.

The term “taxon” is intended to mean a group of microorganisms having ataxonomic unit. A taxon may be a family, a genus, a set of genre, aspecies, a set of species or a subspecies. By way of indication, mentionmay be made of enterobacteria, Klebsiella, Escherichia, Enterobacter,Citrobacter, Serratia, KESC (Klebsiella, Enterobacter, Serratia,Citrobacter), Proteeae, Proteus, Morganella, Pseudomonas,Staphylococcus, Streptococcus, Enterococcus, Candida, Escherichia coli,Escherichia coli 0157:H7, Klebsiella pneumoniae, Citrobacter freundii,Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus,coagulase-negative staphylocoque, Candida albicans, Candida glabrata,Candida krusei, Candida lusitaniae.

The term “antimicrobial” is intended to mean any compound capable ofpreventing or slowing down the growth of a microorganism. This compoundmay be an antibiotic or an antifungal.

The term “antibiotic” is intended to mean any compound capable ofpreventing or slowing down the growth of a bacterium. By way ofindication, mention may in particular be made of the antibioticscefotaxime, ceftazidime, ceftriaxone, cefpodoxime, aztreonam,vancomycin, tobramycin and ciprofloxacin.

The term “antifungal” is intended to mean any compound capable ofpreventing or slowing down the growth of a yeast or of a mould. By wayof indication, mention may in particular be made of amphotericin B,fluconazole, itraconazole, voriconazole and cycloheximide.

According to a preferred embodiment of the invention, when theantibiotic is

-   -   cefotaxime, the cefotaxime concentration in the medium is        preferably between 0.25 and 8 mg/l, preferably between 1 and 2        mg/l;    -   ceftazidime, the ceftazidime concentration in the medium is        preferably between 0.25 and 8 mg/l, preferably between 2 and 2.5        mg/l;    -   ceftriaxone, the ceftriaxone concentration in the medium is        preferably between 0.25 and 8 mg/l, preferably between 1 and 2.5        mg/l;    -   cefpodoxime, the cefpodoxime concentration in the medium is        preferably between 0.1 and 32 mg/l, preferably between 0.75 and        10 mg/l, and even more preferably between 1 and 6 mg/l;    -   aztreonam, the aztreonam concentration in the medium is        preferably between 0.1 and 8 mg/l, preferably between 0.75 and        1.5 mg/l.

According to a specific embodiment of the invention, the mediumcomprises a combination of at least two antibiotics. Preferably, thecombination of at least two antibiotics comprises cefotaxime andceftazidime.

Irrespective of the embodiment of the invention, the medium may alsocomprise a dye. By way of indication of a dye, mention may be made ofEvans blue, neutral red, sheep blood, horse blood, and opacifier such astitanium oxide, nitroaniline, malachite green, brilliant green, etc.

All the media may also comprise, in order to increase their sensitivity:

-   -   at least one antimicrobial that is active against gram-positive        bacteria, such as in particular linezolide or vancomycin;    -   at least one antimicrobial that is active against yeasts, such        as in particular voriconazole or amphotericin B.

In this respect, the invention relates to the use of a combination oftwo culture media for distinguishing at least three groups ofmicroorganisms in a biological sample, comprising:

-   -   a first group of microorganisms, belonging to a first taxon of        microorganisms and comprising at least a first mechanism of        resistance to a first treatment;    -   a second group of microorganisms, belonging to a second taxon of        microorganisms and comprising at least a second mechanism of        resistance to a second treatment;    -   a third group of microorganisms, that are not resistant to said        first and second treatments,        said combination of two culture media comprising:    -   a. at least a first substrate for detecting at least a first        enzymatic or metabolic activity of said first group of        microorganisms;    -   b. at least two markers for differentiating the first group of        microorganisms and the second group of microorganisms;    -   c. at least one antimicrobial that is active on said third group        of microorganisms.

Preferably, the medium comprises at least two markers fordifferentiating the first group of microorganisms and the second groupof microorganisms, at least one of which is an inhibitor of said firstor said second mechanism of resistance. The medium may also comprise atleast two markers for differentiating the first group of microorganismsand the second group of microorganisms, which are each an inhibitor ofsaid first or said second mechanism of resistance.

This first embodiment of the invention makes it possible to distinguish,in the same sample, a first and a second group comprising variousspecies or various taxons of microorganisms, each of the two groupsbeing resistant to a different treatment.

This embodiment of the invention thus makes it possible, for example, todistinguish, in the same sample, a first group comprising E. coli ESBLbacteria and a second group comprising KESC HL Case bacteria. In thisspecific case, the combination of two media may be the following:

-   -   At least a first substrate which makes it possible to        identify E. coli bacteria, for example a glucuronidase        substrate, such as 6-chloro-3-indolyl-β-D-glucuronide, or a        galactosidase substrate, such as        5-bromo-6-chloro-3-indolyl-β-D-galactoside;    -   a first identification marker which is a glucosidase substrate,        such as 5-bromo-4-chloro-3-indolyl-β-D-glucoside, which makes it        possible to identify KESC bacteria;    -   a second identification marker which is a resistance mechanism        inhibitor, such as cloxacillin;    -   at least one antimicrobial which is an antibiotic, such as        cefpodoxime.

Reference is made to a combination of 2 media, i.e. a first medium maycomprise:

-   -   a first substrate which makes it possible to identify E. coli,        for example a glucuronidase substrate, such as        6-chloro-3-indolyl-β-D-glucuronide or a galactosidase substrate,        such as 5-bromo-6-chloro-3-indolyl-β-D-galactoside;    -   a first marker which is a resistance mechanism inhibitor, such        as cloxacillin;    -   an antimicrobial which is an antibiotic, such as cefpodoxime,        or the second medium comprises:    -   a first substrate which makes it possible to identify E coli,        for example a glucuronidase substrate, such as        6-chloro-3-indolyl-β-D-glucuronide, or a galactosidase        substrate, such as 5-bromo-6-chloro-3-indolyl-β-D-galactoside;    -   a first marker which is a glucosidase substrate, such as        5-bromo-4-chloro-3-indolyl-β-D-glucoside, which makes it        possible to identify KESC bacteria;    -   an antimicrobial which is an antibiotic, such as ceftazidime.

Another alternative would be to use a first medium comprising:

-   -   a first substrate which makes it possible to identify E. coli,        for example a glucuronidase substrate, such as        6-chloro-3-indolyl-β-D-glucuronide, or a galactosidase        substrate, such as 5-bromo-6-chloro-3-indolyl-β-D-galactoside;    -   a first marker which is a glucosidase substrate, such as        5-bromo-4-chloro-3-indolyl-β-D-glucoside, which makes it        possible to identify KESC bacteria;    -   an antimicrobial which is an antibiotic, such as aztreonam,        while the second medium comprises:    -   a first substrate which makes it possible to identify E. coli,        for example a glucuronidase substrate, such as        6-chloro-3-indolyl-β-D-glucuronide, or a galactosidase        substrate, such as 5-bromo-6-chloro-3-indolyl-β-D-galactoside;    -   a first marker which is a resistance mechanism inhibitor, such        as clavulanic acid;    -   an antimicrobial which is an antibiotic, such as ceftazidime.

Those skilled in the art will choose each medium so as to systematicallyobtain a combination according to the invention. The antimicrobial thatis active on said third group is present in the two media. Those skilledin the art may in particular use a biplate, for readily comparing thetwo media on which the same biological sample has been deposited.

In this respect, the invention also relates to a biplate comprising acombination of two culture media, said combination comprising:

-   -   a. at least a first substrate for detecting at least a first        enzymatic or metabolic activity of said first group of        microorganisms;    -   b. at least two markers for differentiating the first group of        microorganisms and the second group of microorganisms;    -   c. at least one antimicrobial that is active on said third group        of microorganisms, said antimicrobial being present on each side        of the biplate.

This first embodiment of the invention is not limited to distinguishingbetween 3 groups of microorganisms, but may make it possible todistinguish between 4, 5 or even more groups of microorganisms. It isthen necessary to add additional identification markers to the medium ormedia, in order to discriminate between the various groups.

In this respect, the invention also relates to a biplate comprising acombination of two culture media, said combination comprising:

-   for the first medium:    -   a first substrate which makes it possible to identify E. coli,        for example a glucuronidase substrate such as        6-chloro-3-indolyl-β-D-glucuronide, or a galactosidase        substrate, such as 5-bromo-6-chloro-3-indolyl-β-D-galactoside,        or a substrate which makes it possible to identify Proteeae, for        example tryptophan;    -   a first identification marker which is a glucosidase substrate,        such as 5-bromo-4-chloro-3-indolyl-β-D-glucoside;    -   a second identification marker which is a resistance inhibitor,        such as cloxacillin;    -   at least one antimicrobial which is an antibiotic, such as        cefpodoxime;-   for the second medium:    -   a first substrate which makes it possible to identify        enterococci, for example a glucosidase substrate, such as        5-bromo-4-chloro-3-indolyl-β-D-glucoside;    -   a first identification marker which is another glucosidase        substrate, such as methyl-α-glucoside;    -   at least one antimicrobial which is an antibiotic, such as        vancomycin.

The invention also relates to the use of a culture medium fordistinguishing at least 3 groups of microorganisms in a biologicalsample, comprising:

-   -   a first group of microorganisms, belonging to a first taxon of        microorganisms and comprising at least one mechanism of        resistance to a treatment;    -   a second group of microorganisms, belonging to a second taxon of        microorganisms, different than said first taxon, but comprising        at least one mechanism of resistance to a treatment, identical        to that of the first group;    -   a third group of microorganisms, that are not resistant to said        treatment,

said culture medium comprising:

-   -   a. at least a first substrate for detecting at least a first        enzymatic or metabolic activity of said first group of        microorganisms;    -   b. at least one marker for differentiating the first group of        microorganisms and the second group of microorganisms, said        marker being a substrate for detecting at least one enzymatic or        metabolic activity of said second group of microorganisms;    -   c. at least one antimicrobial that is active on said third group        of microorganisms.

This second embodiment of the invention makes it possible todistinguish, in the same sample, a first and a second group comprisingvarious species or various taxons of microorganisms, but each of the twogroups being resistant to the same treatment. In this specificembodiment of the invention, it is not necessary to use a combination ofmedia, since a single medium comprising the characteristics as definedabove is sufficient.

This embodiment of the invention thus makes it possible, for example, todistinguish, in the same sample, a first group comprising E coli ESBLbacteria and a second group comprising KESC ESBL bacteria.

In this respect, the invention relates to a culture medium comprising:

-   -   a first substrate for detecting a beta-glucosidase enzymatic        activity, preferably 5-bromo-4-chloro-3-indolyl-β-D-glucoside,        at a concentration of between 25 and 500 mg/l, preferably        between 40 and 150 mg/l;    -   a second substrate for detecting methyl-alpha-glucoside        metabolism, in the presence of a colored indicator, preferably        neutral red, at a concentration of between 2 and 100 mg/l,        preferably between 4 and 50 mg/l;    -   an antimicrobial which is an antibiotic, preferably vancomycin,        at a concentration of between 0.5 and 128 mg/l, preferably        between 2 and 32 mg/l.

When the antibiotic is vancomycin, this medium is preferably used fordistinguishing:

-   -   a first group of enterococcal bacteria developing an acquired        resistance against vancomycin;    -   a second group of enterococcal bacteria developing a natural        resistance against vancomycin;    -   a third group of enterococcal bacteria that are not resistant to        vancomycin.

The invention also relates to a culture medium comprising:

-   -   a first substrate for detecting a hexosaminidase enzymatic        activity, preferably        5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-glucosaminide, and a        concentration between 25 and 500 mg/l, preferably between 40 and        150 mg/l;    -   a second substrate for detecting a beta-glucosidase activity,        preferably 6-chloro-3-indolyl-β-D-glucoside, at a concentration        of between 25 and 500 mg/l, preferably between 40 and 200 mg/l;    -   an antimicrobial which is an antifungal, which is preferably        amphotericin B (amphoB), at a concentration of between 0.5 and        64 mg/l, preferably between 1 and 16 mg/l, even more preferably        between 1 and 8 mg/l.

When the antifungal is amphotericin B, this medium is preferably used todistinguish:

-   -   a first group of yeasts comprising Candida albicans developing a        resistance to amphoB;    -   a second group of yeasts comprising Candida tropicalis and/or C.        lusitaniae and/or C. kefyr, developing a resistance to amphoB;    -   a third group of yeasts that are not resistant to amphoB.

The invention also relates to a culture medium comprising:

-   -   a first substrate for detecting a hexosaminidase enzymatic        activity, preferably        5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-glucosaminide, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 150 mg/l;    -   a second substrate for detecting a phosphatase activity,        preferably 5-bromo-6-chloro-3-indolylphosphate, at a        concentration of between 25 and 750 mg/l, preferably between 40        and 200 mg/l;    -   an antimicrobial which is an antifungal, which is preferably        amphoB, at a concentration of between 0.5 and 64 mg/l,        preferably between 1 and 16 mg/l, even more preferably between 1        and 8 mg/l.

When the antifungal is amphotericin B, this medium is preferably used todistinguish:

-   -   a first group of yeasts comprising Candida albicans developing a        resistance to amphoB;    -   a second group of yeasts comprising Candida tropicalis and/or C.        glabrata and/or C. krusei, developing a resistance to amphoB;    -   a third group of yeasts that are not resistant to amphoB.

The invention also relates to a culture medium comprising:

-   -   a first substrate for detecting the hexosaminidase enzymatic        activity of said first group, preferably        5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-glucosaminide, at a        concentration of between 25 and 500 mg/l, preferably of between        40 and 150 mg/l;    -   a second substrate for detecting the beta-glucosidase activity        of said second group, preferably        6-chloro-3-indolyl-β-D-glucoside, at a concentration of between        25 and 500 mg/l, preferably between 40 and 200 mg/l;    -   an antimicrobial which is an antifungal, preferably fluconazole,        at a concentration of between 1 and 256 mg/l, preferably between        2 and 128 mg/l, even more preferably between 8 and 64 mg/l.

When the antifungal is fluconazole, this medium is preferably used todistinguish:

-   -   a first group of yeasts comprising Candida albicans developing a        resistance to fluconazole;    -   a second group of yeasts, comprising Candida tropicalis        and/or C. lusitaniae and/or C. kefyr, developing a resistance to        fluconazole;    -   a third group of yeasts that are not resistant to fluconazole.

The invention also relates to a culture medium comprising:

-   -   a first substrate for detecting a hexosaminidase enzymatic        activity, preferably        5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-glucosaminide, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 150 mg/l;    -   a second substrate for detecting a phosphatase activity,        preferably 5-bromo-6-chloro-3-indolylphosphate, at a        concentration of between 25 and 750 mg/l, preferably between 40        and 200 mg/l;    -   an antimicrobial which is an antifungal, which is preferably        fluconazole, at a concentration of between 1 and 256 mg/l,        preferably between 2 and 128 mg/l, even more preferably between        8 and 64 mg/l.

When the antifungal is fluconazole, this medium is preferably used todistinguish:

-   -   a first group of yeasts comprising Candida albicans developing a        resistance to fluconazole;    -   a second group of yeasts, comprising Candida tropicalis        and/or C. glabrata and/or C. krusei, developing a resistance to        fluconazole;    -   a third group of yeasts that are not resistant to fluconazole.

The invention also relates to a culture medium comprising:

-   -   a first substrate for detecting a beta-glucuronidase enzymatic        activity, preferably 6-chloro-3-indolyl-β-D-glucuronide, at a        concentration of between 25 and 750 mg/l, preferably between 40        and 300 mg/l, or a beta-galactosidase enzymatic activity,        preferably 5-bromo-6-chloro-3-indolyl-β-D-galactoside, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 150 mg/l;    -   a second substrate for detecting a beta-glucosidase activity,        preferably 5-bromo-4-chloro-3-indolyl-β-D-glucoside, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 250 mg/l, or a tryptophanase or desaminase activity,        preferably tryptophan, at a concentration of between 50 and 5000        mg/l, preferably between 250 and 2000 mg/l;    -   an antimicrobial which is an antibiotic, preferably ceftazidime.        The ceftazidime concentration in the medium is then preferably        between 0.25 and 8 mg/l, preferably between 2 and 2.5 mg/l.

When the antibiotic is ceftazidime, this medium is preferably used todistinguish:

-   -   a first group of E. coli ESBL or HL Case bacteria;    -   a second group of KESC ESBL or HL Case bacteria;    -   a third group of bacteria that are not resistant to        beta-lactamines and to cephalosporins.

The invention also relates to a culture medium comprising:

-   -   a first substrate for detecting a beta-glucuronidase or        beta-galactosidase enzymatic activity, preferably        6-chloro-3-indolyl-β-D-glucuronide, at a concentration of        between 25 and 750 mg/l, preferably between 40 and 300 mg/l, or        5-bromo-6-chloro-3-indolyl-β-D-galactoside, at a concentration        of between 25 and 500 mg/l, preferably between 40 and 150 mg/l;    -   a second substrate for detecting a beta-glucosidase activity,        preferably 5-bromo-4-chloro-3-indolyl-β-D-glucoside, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 250 mg/l, or a tryptophanase or desaminase activity,        preferably tryptophan, at a concentration of between 50 and 5000        mg/l, preferably between 250 and 2000 mg/l;    -   an antimicrobial which is an antibiotic, preferably cefpodoxime,        at a concentration of between 0.5 and 32 mg/l, preferably        between 0.75 and 10 mg/l, and even more preferably between 1 and        6 mg/l, and cloxacillin, at a concentration of between 10 and        2000 mg/l, preferably between 50 and 500 mg/l.

When the antibiotic is cefpodoxime, this medium is preferably used todistinguish:

-   -   a first group of E. coli ESBL bacteria;    -   a second group of KESC ESBL bacteria;    -   a third group of bacteria that are not resistant to        beta-lactamines.

The invention also relates to a culture medium comprising:

-   -   a first substrate for detecting a beta-glucuronidase enzymatic        activity, preferably 6-chloro-3-indolyl-β-D-glucuronide, at a        concentration of between 25 and 750 mg/l, preferably between 40        and 300 mg/l, or a beta-galactosidase enzymatic activity,        preferably 5-bromo-6-chloro-3-indolyl-β-D-galactoside, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 150 mg/l;    -   a second substrate for detecting a beta-glucosidase activity,        preferably 5-bromo-4-chloro-3-indolyl-β-D-glucoside, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 250 mg/l, or a tryptophanase or desaminase activity,        preferably tryptophan, at a concentration of between 50 and 5000        mg/l, preferably between 250 and 2000 mg/l;    -   a combination comprising an antimicrobial and a resistance        inhibitor. Preferably, this combination comprises ceftriaxone at        a concentration of between 0.25 and 8 mg/l, preferably between 1        and 2.5 mg/l, and clavulanic acid at a concentration of between        0.05 and 32 mg/l, preferably between 0.1 and 8 mg/l, and even        more preferably between 0.5 and 6 mg/l.

This medium is preferably used to distinguish:

-   -   a first group of E. coli HL Case bacteria;    -   a second group of KESC HL Case bacteria;    -   a third group of bacteria that are not resistant to        cephalosporins.

The invention also relates to a culture medium comprising:

-   -   a first substrate for detecting a beta-glucuronidase enzymatic        activity, preferably 6-chloro-3-indolyl-β-D-glucuronide, at a        concentration of between 25 and 750 mg/l, preferably between 40        and 300 mg/l, or a beta-galactosidase enzymatic activity,        preferably 5-bromo-6-chloro-3-indolyl-β-D-galactoside, at a        concentration between 25 and 500 mg/l, preferably between 40 and        150 mg/l;    -   a second substrate for detecting a beta-glucosidase activity,        preferably 5-bromo-4-chloro-3-indolyl-β-D-glucoside, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 250 mg/l, or a tryptophanase or desaminase activity,        preferably tryptophan, at a concentration of between 50 and 5000        mg/l, preferably between 250 and 2000 mg/l;    -   two antimicrobials, which are preferably two antibiotics,        preferably cefpodoxime at a concentration of between 0.1 and 32        mg/l, preferably between 0.25 and 10 mg/l, and even more        preferably between 0.5 and 4 mg/l, and aztreonam at a        concentration of between 0.1 and 8 mg/l, preferably between 0.5        and 1.5 mg/l.

This medium is preferably used to distinguish:

-   -   a first group of E. coli ESBL or HL Case bacteria;    -   a second group of KESC ESBL or HL Case bacteria;    -   a third group of bacteria that are not resistant to        beta-lactamines and/or to cephalosporins.

The invention also relates to a culture medium comprising:

-   -   at least a first substrate for detecting alpha-glucoside        metabolism or for detecting alpha-glucosidase activity,        preferably methyl-α-glucoside, at a concentration of between 1        and 50 g/l, preferably between 5 and 20 g/l, or        5-bromo-4-chloro-3-indolyl-α-D-glucoside, at a concentration of        between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or        5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucoside, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 250 mg/l;    -   at least a second substrate for detecting a second activity        different than alpha-glucoside metabolism or the        alpha-glucosidase activity, preferably        5-bromo-4-chloro-3-indolyl-β-D-glucoside, at a concentration of        between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or        6-chloro-3-indolyl-β-D-glucoside, at a concentration of between        25 and 500 mg/l, preferably between 40 and 250 mg/l, or        alizarine-β-D-galactoside, at a concentration of between 10 and        500 mg/l, preferably between 20 and 250 mg/l, or        5-bromo-6-chloro-3-indolyl-β-D-glucoside, at a concentration of        between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or        5-bromo-6-chloro-3-indolyl-β-D-galactoside, at a concentration        of between 25 and 500 mg/l, preferably between 40 and 250 mg/l,        or 6-chloro-3-indolyl-β-D-galactoside, at a concentration of        between 25 and 500 mg/l, preferably between 40 and 250 mg/l.        Preferably, this second substrate makes it possible to detect a        beta-glucosidase or beta-galactosidase activity; and    -   at least one antimicrobial, preferably an antibiotic, such as        vancomycin, at a concentration of between 0.5 and 128 mg/l,        preferably between 2 and 32 mg/l.

When the antibiotic is vancomycin, this medium is preferably used todistinguish:

-   -   a first group of vancomycin-resistant microorganisms, comprising        Enterococcus faecalis and Enterococcus faecium;    -   a second group of vancomycin-resistant microorganisms,        comprising Enterococcus casseliflavus and Enterococcus        gallinarum;    -   a third group of microorganisms that are not resistant to        vancomycin.

In this case, the first substrate is preferably methyl-α-glucoside, thesecond substrate is preferably 5-bromo-4-chloro-3-indolyl-β-D-glucosideor 6-chloro-3-indolyl-β-D-glucoside, and the antimicrobial is preferablyvancomycin.

This medium is also preferably used to distinguish:

-   -   a first group of vancomycin-resistant microorganisms, comprising        Enterococcus faecalis;    -   a second group of vancomycin-resistant microorganisms,        comprising Enterococcus faecium;    -   a third group of microorganisms that are not resistant to        vancomycin or that express a natural resistance (E.        casseliflavus and E. gallinarum).

In this case, the first substrate is preferably5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucoside or5-bromo-4-chloro-3-indolyl-α-D-glucoside, the second substrate ispreferably 6-chloro-3-indolyl-β-D-glucoside or alizarine-β-D-galactosideor 5 bromo-6-chloro-3-indolyl-β-D-glucoside or5-bromo-6-chloro-3-indolyl-β-D-galactoside or6-chloro-3-indolyl-β-D-galactoside, and the antimicrobial is preferablyvancomycin.

This medium is also preferably used to distinguish:

-   -   a first group of vancomycin-resistant microorganisms, comprising        Enterococcus faecalis and Enterococcus faecium;    -   a second group of microorganisms, comprising Staphylococcus        aureus, that are intermediately resistant or resistant to        vancomycin;    -   a third group of microorganisms that are not resistant to        vancomycin.

In this case, the first substrate is preferably5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucoside or5-bromo-4-chloro-3-indolyl-β-D-glucoside, the second substrate ispreferably 6-chloro-3-indolyl-β-D-glucoside or5-bromo-6-chloro-3-indolyl-β-D-glucoside, and the antimicrobial ispreferably vancomycin.

The table below makes it possible to distinguish the appropriatecombinations of substrates and antimicrobial according to the speciesthat it is desired to detect:

1st group of 2nd group of 3rd group of microorganisms microorganismsmicroorganisms 1st substrate 2nd substrate Antimicrobial E. faecalis andE. casseliflavus Microorganisms Methyl-α-glucoside 5-bromo- vancomycinE. faecium, and E. that are not 4-chloro- resistant to gallinarum,resistant to 3-indolyl- vancomycin resistant to vancomycin β-D-glucosideor vancomycin 6-chloro- 3-indolyl- β-D-glucoside E. faecalis, E.faecium, Microorganisms 5-bromo-4-chloro- 6-chloro- vancomycin resistantto resistant to that are not 3-indolyl-N-methyl- 3-indolyl- vancomycinvancomycin resistant to α-D-glucoside or β-D-glucoside or vancomycin or5-bromo-4-chloro- alizarine- that express a 3-indolyl- β-D-galactosidenatural resistance α-D-glucoside or 5-bromo- (E. casseliflavus6-chloro-3- and E. indolyl-β- gallinarum) D-glucoside or 5-bromo-6-chloro-3-indolyl- β-D-galactoside or 6-chloro- 3-indolyl-β-D-galactoside E. faecalis and S. aureus, Microorganisms5-bromo-4-chloro- 6-chloro- vancomycin E. faecium, resistant to that arenot 3-indolyl-N-methyl- 3-indolyl- resistant to vancomycin resistant toα-D-glucoside or β-D-glucoside or vancomycin vancomycin5-bromo-4-chloro- 5-bromo- 3-indolyl- 6-chloro-3- α-D-glucosideindolyl-β-D- glucosideIt may be relevant to also adjust the vancomycin concentration,preferably to between 0.5 and 12 mg/l.

The invention also relates to a culture medium comprising:

-   -   an antimicrobial, preferably an antibiotic such as cloxacillin;    -   a resistance mechanism inhibitor, such as preferably a        3rd-generation cephalosporin chosen from cefotaxime,        ceftazidime, cefpodoxime and ceftriaxone.

This medium is also preferably used to detect ESBL bacteria.

The second embodiment of the invention is not limited to distinguishing3 groups of microorganisms, but can make it possible to distinguish 4, 5or even more groups of microorganisms. It is then necessary to add, tothe medium, markers for identification between the various groups.

In this respect, the invention also relates to a culture mediumcomprising:

-   -   a first substrate for detecting a beta-glucuronidase enzymatic        activity, preferably 6-chloro-3-indolyl-β-D-glucuronide, at a        concentration of between 25 and 750 mg/l, preferably between 40        and 300 mg/l, or a beta-galactosidase enzymatic activity,        preferably 5-bromo-6-chloro-3-indolyl-β-D-galactoside, at a        concentration of between 25 and 500 mg/l, preferably of between        40 and 150 mg/l;    -   a second substrate for detecting a beta-glucosidase, preferably        5-bromo-4-chloro-3-indolyl-β-D-glucoside, at a concentration of        between 25 and 500 mg/l, preferably between 40 and 250 mg/l;    -   a combination of antimicrobials, preferably a combination of        antibiotics such as        -   cefpodoxime at a concentration of between 0.5 and 32 mg/l,            preferably between 0.75 and 10 mg/l, and even more            preferably between 1 and 6 mg/l;        -   cloxacillin at a concentration of between 10 and 2000 mg/l,            preferably between 50 and 500 mg/l;        -   vancomycin at a concentration of between 0.5 and 128 mg/l,            preferably between 2 and 32 mg/l; and        -   amphoB at a concentration of between 0.5 and 64 mg/l,            preferably between 1 and 16 mg/l, even more preferably            between 1 and 8 mg/l;    -   a third substrate for detecting a desaminase activity, such as        histidine, phenylalanine, tryptophan or tyrosine, at a        concentration of between 50 and 5000 mg/l, preferably between        250 and 2000 mg/l.

This medium may also comprise a fifth antibiotic, which is cefsulodine,at a concentration of between 0.5 and 64 mg/l, preferably between 1 and16 mg/l.

This medium is preferably used to distinguish:

-   -   a first group of E. coli ESBL bacteria;    -   a second group of KESC ESBL bacteria;    -   a third group of bacteria that are not resistant to        beta-lactamines and/or to cephalosporins;    -   a fourth group of Proteeae ESBL bacteria.

Similarly, the invention also relates to a culture medium comprising:

-   -   at least a first substrate for detecting alpha-glucoside        metabolism or for detecting alpha-glucosidase activity,        preferably 5-bromo-4-chloro-3-indolyl-α-D-glucoside, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 250 mg/l, or        5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucoside, at a        concentration of between 25 and 500 mg/l, preferably between 40        and 250 mg/l;    -   at least a second substrate for detecting a second activity        different than alpha-glucoside metabolism or the        alpha-glucosidase activity, preferably        6-chloro-3-indolyl-β-D-glucoside, at a concentration of between        25 and 500 mg/l, preferably between 40 and 250 mg/l, or        6-chloro-3-indolyl-β-D-galactoside, at a concentration of        between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or        alizarine-β-galactoside, at a concentration of between 10 and        500 mg/l, preferably between 20 and 250 mg/l, or        5-bromo-6-chloro-3-indolyl-β-D-glucoside, at a concentration of        between 25 and 500 mg/l, preferably between 40 and 250 mg/l, or        5-bromo-6-chloro-3-indolyl-β-D-galactoside, at a concentration        of between 25 and 500 mg/l, preferably between 40 and 250 mg/l,        or 6-chloro-3-indolyl-β-D-galactoside, at a concentration of        between 25 and 500 mg/l, preferably between 40 and 250 mg/l.        Preferably, this second substrate makes it possible to detect a        beta-glucosidase or beta-galactosidase activity; and    -   a combination of antimicrobials, preferably a combination of        antibiotics such as        -   vancomycin at a concentration of between 0.5 and 128 mg/l,            preferably between 2 and 32 mg/l;        -   aztreonam at a concentration of between 1 and 150 mg/l,            preferably between 4 and 60 mg/l;        -   colistine at a concentration of between 1 and 100 mg/l,            preferably between 2 and 20 mg/l;        -   amphotericin B at a concentration of between 0.5 and 50            mg/l, preferably between 1 and 15 mg/l.

When one of the antibiotics is vancomycin, this medium is preferablyused to distinguish:

-   -   a first group of vancomycin-resistant microorganisms, comprising        Enterococcus faecium,    -   a second group of vancomycin-resistant microorganisms,        comprising Enterococcus faecalis,    -   a third group of microorganisms that are not resistant to        vancomycin.

This medium is also preferably used to distinguish:

-   -   a first group of vancomycin-resistant microorganisms, comprising        Enterococcus faecium,    -   a second group of microorganisms, comprising Staphylococcus        aureus, that are intermediately resistant or resistant to        vancomycin;    -   a third group of microorganisms that are not resistant to        vancomycin.

The combinations of substrates according to the groups of microorganismsthat it is desired to identify are presented, for example, in the tableon page 21. By using an appropriate combination of antimicrobials, it ispossible to distinguish not only three groups of microorganisms, butalso 4, 5 or even more groups of microorganisms.

The invention also relates to the use of a combination of two culturemedia for distinguishing at least 3 groups of microorganisms in abiological sample, comprising:

-   -   a first group of microorganisms, belonging to a first species of        microorganisms and comprising a first mechanism of resistance to        a first treatment;    -   a second group of microorganisms, belonging to a species of        microorganisms that is identical to that of said first group of        microorganisms, but comprising a second mechanism of resistance        to a second treatment, different than that of said first group;    -   a third group of microorganisms that are not resistant to said        first and second treatment,

said combination of two culture media comprising:

-   -   a. at least a first substrate for detecting at least a first        enzymatic or metabolic activity of said first group of        microorganisms;    -   b. at least one marker for differentiating the first group of        microorganisms and the second group of microorganisms, said        marker being an inhibitor of the mechanism of resistance to the        first treatment and/or to the second treatment;    -   c. at least one antimicrobial that is active on said third group        of microorganisms.

This third embodiment of the invention makes it possible to distinguish,in the same sample, a first and a second group comprising the samespecies of microorganisms or the same taxon, each of the two groupsbeing resistant to different treatments.

This embodiment of the embodiment thus makes it possible, for example,to distinguish, in the same sample, a first group comprising E. coliESBL bacteria, and a second group comprising E. coli HL Case bacteria.In this specific case, the combination of two media may be thefollowing:

-   -   at least a first substrate which makes it possible to        identify E. coli, for example a beta-glucuronidase substrate,        such as 6-chloro-3-indolyl-β-D-glucuronide, or a        beta-glucosidase substrate, such as        5-bromo-6-chloro-3-indolyl-β-D-galactoside;    -   a first identification marker which is a resistant mechanism        inhibitor, preferably cloxacillin;    -   a second identification marker which is another resistance        mechanism inhibitor, preferably clavulanic acid;    -   at least one antimicrobial which is cefpodoxime.

Reference is made to a combination of 2 media, i.e. a first medium maycomprise:

-   -   a first substrate which makes it possible to identify E. coli,        which is 6-chloro-3-indolyl-β-D-glucuronide;    -   a first marker which is a resistance mechanism inhibitor,        preferably cloxacillin;    -   an antimicrobial, preferably an antibiotic, preferably        cefpodoxime,

while the second medium comprises:

-   -   a first substrate which makes it possible to identify E. coli,        such as 6-chloro-3-indolyl-β-D-glucuronide;    -   a second marker which is another resistance mechanism inhibitor,        preferably clavulanic acid;    -   an antimicrobial, preferably an antibiotic, preferably        cefpodoxime.

Another alternative would be to use a first medium comprising:

-   -   a first substrate which makes it possible to identify E. coli,        which is 5-bromo-6-chloro-3-indolyl-β-D-galactoside;    -   a first marker which is a substrate for a desaminase or for a        tryptophanase, preferably tryptophan;    -   an antimicrobial, preferably an antibiotic, preferably        cefpodoxime,

or the second medium comprises:

-   -   a first substrate which makes it possible to identify E. coli,        which is 6-chloro-3-indolyl-β-D-glucuronide;    -   a second marker which is a resistance mechanism inhibitor,        preferably clavulanic acid;    -   an antimicrobial, preferably an antibiotic, which is preferably        ceftazidime.

Those skilled in the art will choose each medium in such a way as tosystematically obtain a combination according to the invention. Theantimicrobial that is active on said third group is present in the twomedia. Those skilled in the art may in particular use a biplate, whichmakes it possible to readily compare the two media on which the samebiological sample would have been deposited.

In this respect, the invention also relates to a biplate comprising acombination of two culture media, said combination comprising:

-   -   a. at least a first substrate for detecting at least a first        enzymatic or metabolic activity of said first group of        microorganisms;    -   b. at least one marker for differentiating the first group of        microorganisms and the second group of microorganisms, said        marker being an inhibitor of the mechanism of resistance to the        first treatment and/or to the second treatment;    -   c. at least one antimicrobial that is active on said third group        of microorganisms.

This third embodiment of the invention is not limited to distinguishing3 groups of microorganisms, but may make it possible to distinguish 4, 5or even more groups of microorganisms. It is then necessary to addadditional identification markers to the medium, in order todiscriminate the various groups.

In this respect, the invention relates to a biplate comprising acombination of two culture media, said combination comprising:

-   for the first medium,    -   a first substrate which makes it possible to identify E. coli,        for example a glucuronidase substrate, such as        6-chloro-3-indolyl-β-D-glucuronide, or a galactosidase        substrate, such as 5-bromo-6-chloro-3 -indolyl-β-D-galactoside;    -   a second substrate which makes it possible to identify Proteeae,        for example a substrate for a desaminase or for a tryptophanase,        such as tryptophan;    -   a third substrate which makes it possible to identify the KESC        group, for example a glucosidase substrate, such as        5-bromo-4-chloro-3-indolyl-β-D-glucoside;    -   a marker for differentiating between ESBL strains and HL Case        strains, preferably a resistance inhibitor, preferably        clavulanic acid;    -   at least one antimicrobial which is preferably an antibiotic,        preferably ceftazidime;-   for the second medium:    -   a first substrate which makes it possible to identify E. coli,        for example a glucuronidase substrate, such as in particular        6-chloro-3-indolyl-β-D-glucuronide, or a galactosidase        substrate, such as in particular        5-bromo-6-chloro-3-indolyl-β-D-galactoside;    -   a second substrate which makes it possible to identify Proteeae,        for example a substrate for a desaminase or for a tryptophanase,        such as tryptophan;    -   a third substrate which makes it possible to identify the KESC        group, for example a glucosidase substrate, such as in        particular 5-bromo-4-chloro-3-indolyl-β-D-glucoside;    -   at least one antimicrobial, preferably an antibiotic, preferably        ceftriaxone.

The examples below are given by way of explanation and are no waylimiting in nature. They will make it possible to understand theinvention more fully.

EXAMPLE 1

The example below is based on the phenotypic detection of ESBLs usingthe reduction of susceptibility of these strains to antibiotics andtheir sensitivity to combinations with β-lactamases inhibitors. Forthis, a biplate of CPS ID 3 base (chromogenic medium for detectingmicroorganisms in urine, and sold by bioMérieux under the reference43541) with one half-agar containing an antibiotic and one half-agarcontaining an antibiotic/β-lactamases inhibitor combination was used.

1. Choice of Strains

In the context of the manipulations carried out, for evaluating theactivity of the antibiotics active on gram-negative bacilli, variousspecies of enterobacteria (Escherichia coli, Enterobacter aerogenes,Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis) and ofnonfermenting gram-negative bacilli (Pseudomonas aeruginosa), capable ofproducing ESBLs, were used. ESBL-positive strains, high levelcephalosporinase-producing strains (HL Case) and wild-type strains arecompared in the trials.

For the manipulations regarding the antibiotics active on gram-positivebacteria, strains of gram-positive cocci (Staphylococcus aureus,Staphylococcus saprophyticus, Staphylococcus epidermidis, Enterococcusfaecalis, Enterococcus faecium, Streptococcus agalactiae) and ofgram-positive bacilli (Lactobacillus spp) were tested.

For the trials intended to evaluate the activity of the antifungals,various strains of yeasts (Candida albicans, Candida glabrata, Candidatropicalis, Candida krusei, Candida dubliniensis, Saccharomycescerevisiae, Geotrichum capitatum) were used.

2. Preparation of Media

The medium used was a CPS ID3 medium (reference 43541) also comprisingat least one antibiotic and at least one resistance mechanism inhibitor.

The composition of the media tested was the following:

Chromogenic substrate Antibiotic Resistance inhibitor Medium A6-chloro-3-indolyl-β-D-glucuronide Cefotaxime 1 mg/l5-bromo-4-chloro-3-indolyl-β-D-glucoside Tryptophan FeCl₃ Total: 1.73g/l Medium B 6-chloro-3-indolyl-β-D-glucuronide Ceftazidime 1.5 mg/l0.25 mg/l clavulanic acid 5-bromo-4-chloro-3-indolyl-β-D-glucosideTryptophan FeCl₃ Total: 1.73 g/lOsmosed water is added and the whole is homogenized and melted in awaterbath at 100° C. The base medium is dispensed into flasks, thenumber of which corresponds to the total number of media to be testedduring the process. The flasks are then autoclaved for 15 min at 121° C.The media are brought back to and kept molten at 55±3° C. in awaterbath, in order to sterilely add the thermolabile additives(sterilized beforehand by filtration through 0.22 μm). The media arethen poured into biplates 90 mm in diameter (i.e. approximately 9.5ml/half-plate), and left on a flat surface so that they can set. Thesurface of the agars is then dried under a laminar flow hood for 30 min.

3. Inoculation of Media

An inoculum of 0.5 McF is prepared, in physiological saline, from24-hour precultures at 36° C.±2° C. in an aerobic atmosphere on TSAmedium, and then 1 μl of this suspension is transferred into 5 ml ofphysiological saline. In order to obtain a sufficient number of isolatedcolonies, a range of inocula made it possible to determine that theoptimal amount of bacteria to be inoculated was from 10³ to 10⁴ CFU/ml.The inoculation is carried out directly on the two half-agars using asterile swab. The cultures are then incubated at 37° C. in an aerobicatmosphere.

4. Reading of media: the readings are carried out at 18 hours (±30 min),24 h (±1h) and 48 h (±4 h). The density and the size of the colonies,and the appearance, the color and the coloration intensities of the massand of the isolated colonies were observed, according to the followingreading scales 1 to 3: 0: no growth; 0.1: trace of growth; 0.25:colonies of diameter<0.5 mm; 0.5: colonies of 0.5 mm in diameter; 0.75:0.5 mm<diameter<1 mm; 1: colonies of 1 mm in diameter; 1.25: 1mm<diameter<1.5 mm; 1.5: colonies 1.5 mm in diameter; 2: colonies 2 mmin diameter; 3: colonies of diameter>2 mm.

5. Results a) Screening of Antibiotics

The 5 antibiotics recommended by the NCCLS (National Committee forClinical Laboratory Standards) were tested. For each product tested, arange was prepared in order to determine the concentrations that make itpossible to inhibit the wild-type strains of the enterobacteria tested,without affecting the growth of the ESBL-positive strains or the HLCase-positive strains.

The concentrations selected are listed in table III hereinafter.

TABLE III Limiting values of the concentrations of antibiotics thatallow inhibition of the wild- type strains of the enterobacteria tested,without affecting the growth of the ESBL-positive strains under theconditions tested Low value High value Cefotaxime 1 mg/l   2 mg/lCeftazidime 2 mg/l 2.5 mg/l Ceftriaxone 1 mg/l 2.5 mg/l Cefpodoxime 2mg/l  10 mg/l Aztreonam 1 mg/l 1.5 mg/l

The low value corresponds to the minimum concentration of the antibioticrequired to inhibit the wild-type strains of the enterobacteria tested.The high value corresponds to the maximum concentration of theantibiotic that can be used without affecting the growth of theESBL-positive strains tested.

At these concentrations, the separation of the wild-type and resistantstrains is satisfactory, and the expression of the enzymatic activitieson the CPS ID 3 medium is compliant. In addition, it should be notedthat ceftazidime is the only antibiotic tested and used in the detectionof ESBLs which showed an activity on the wild-type strains of P.aeruginosa. A range was prepared in order to define the minimumconcentration for complete inhibition of these strains, and the limitingvalue is 1.5 mg/l.

The addition of antibiotics had no effect on the expression of thebacterial enzymatic activities on the chromogenic medium. The groups ofmicroorganisms were separated and identified both on control CPS ID 3medium and on the media comprising the antibiotics as described above.

b) Screening of β-lactamase inhibitors

Three β-lactamases inhibitors (BLIs), i.e. clavulanic acid, tazobactamand sulbactam, were used. A range was prepared for each one, in thepresence of cefotaxime, in order to determine the optimum concentrationfor inhibiting the ESBL-positive strains without impairing the growth ofthe HL Case strains.

Clavulanic acid appeared to be the most effective BLI in the presence ofcefotaxime. Tazobactam and sulbactam required concentrations of greaterthan 2 mg/l in order to inhibit the ESBL-positive strains, whereasclavulanic acid was more active at much lower concentrations, over abroad operating range (from 0.1 to 8 mg/l when it is used in combinationwith cefotaxime).

Each antibiotic (cefotaxime, ceftazidime, ceftriaxone, cefpodoxime,aztreonam) was tested in the presence of a range of clavulanic acid inorder to define the most effective combinations. The combinationsselected are listed in table IV below.

TABLE IV Selected combinations of antibiotics and of clavulanic acid(CA) that inhibit the ESBL-positive enterobacterial strains tested, butallow the HL Case-positive strains to grow Cefotaxime   2 mg/l + CA  0.1mg/l Ceftazidime 2.5 mg/l + CA   2 mg/l Ceftriaxone   2 mg/l + CA 0.25mg/l Cefpodoxime   9 mg/l + CA 0.25 mg/l Aztreonam   1 mg/l + CA  0.5mg/l

The results obtained using a biplate containing the antibiotic alone onone side and the antibiotic+clavulanic acid combination on the otherside are listed in table V.

Table V Antibiotic alone Antibiotic + clavulanic acid Wild-type E. coliESBL-positive E. coli Pink colonies HL Case-positive E. coli Pinkcolonies Pink colonies Wild-type Proteeae ESBL-positive Proteeae Browncolonies HL Case-positive Proteeae Brown colonies Brown coloniesWild-type KESC ESBL-positive KESC Green colonies HL Case-positive KESCGreen colonies Green colonies

The addition of β-lactamase inhibitors had no effect on the expressionof the bacterial enzymatic activities on the chromogenic medium. Thegroups of microorganisms were separated and identified both on controlCPS ID 3 medium and on the media comprising β-lactamase inhibitors.

c) Combination of antibiotics

Given the relative activities of the antibiotics and of clavulanic acid,the following were combined:

-   -   cefotaxime (antibiotic active on ESBL-positive strains in        combination with the lowest concentration of clavulanic acid),    -   ceftazidime (antibiotic active on wild-type strains of P.        aeruginosa), and    -   clavulanic acid,        so as to be able to inhibit, firstly, the wild-type strains        (including those of pyocyanic bacillus), and the ESBL-positive        strains of the enterobacteria tested by the addition of the BIL.

Such a combination was tested on strains of P. aeruginosa; this speciesis naturally resistant to cefotaxime but sensitive to ceftazidime. When1.5 mg/l of CAZ (ceftazidime), 1 mg/l of CTX (cefotaxime) and 0.25 mg/lof CA (clavulanic acid) were combined, all the wild-type strains and theESBL-positive strains of the enterobacteria tested were inhibited andonly the collection of HL Case strains and the ESBL-positive Paeruginosa strains grew. It involves a biplate with CAZ alone on oneside and CTX plus clavulanic acid on the other.

The addition of these combinations of antibiotics had no effect on theexpression of the bacterial enzymatic activities on the chromogenicmedium. The groups of microorganisms were separated and identified bothon control CPS ID 3 medium and on the media comprising such combinationsof antibiotics.

d). Dye Assay

A medium according to the invention was also employed for use in abiplate, one of the sides containing an antibiotic, and the secondcontaining another antibiotic or a combination of antibiotics. Giventhat the same CPS ID 3 medium base is used on either side, these twosides were differentiated by the presence of a dye.

The dye tested, Evans blue, gives the medium a green color. Thecoloration has made it possible to readily differentiate the 2 sides ofthe biplate without affecting the fertility of the medium, or impairingthe reading of the enzymatic activities of the colonies. After havingproduced a range of Evans blue, added before or after autoclaving, thevalues selected were the following:

-   -   1.5 or 2 mg/l if the dye is added after autoclaving.    -   between 2 and 5 mg/l if it is added before autoclaving.

e). Inhibition of Gram-positive Bacteria

ESBLs are a mechanism of resistance to β-lactamines that is found onlyin gram-negative bacilli; it is therefore advisable to inhibit thegram-positive bacteria via the medium. Two antibiotics, linezolide andvancomycin, were used in the medium according to the invention for thepurpose of inhibiting the sensitive gram-positive bacteria.

For vancomycin, under the conditions tested, a concentration between 2and 32 mg/l, and in particular 2 to 5 mg/l, makes it possible to inhibitthe sensitive gram-positive bacteria without interfering with thedetection of the ESBL bacteria.

For linezolide, under the conditions tested, a concentration between 2and 64 mg/l, and in particular 4 to 16 mg/l, makes it possible toinhibit the sensitive gram-positive bacteria without interfering withthe detection of the ESBL bacteria.

The inhibition of the gram-positive bacteria made it possible to improvethe detection of the ESBL bacteria in polymicrobial samples and thespecificity of their coloration.

f) Inhibition of Yeasts

A medium according to the invention can also comprise antifungals inorder to inhibit the possible presence of yeasts which could grow on themedium and which could impair microorganism growth.

Two antifungals were therefore tested: voriconazole and amphotericin B.

For amphotericin B, under the conditions tested, a concentration between1 and 32 mg/l, and in particular 2 to 8 mg/l, makes it possible toinhibit the sensitive yeasts without interfering with the detection ofthe ESBL bacteria.

For voriconazole, under the conditions tested, a concentration ofbetween 1 and 64 mg/l, and in particular 4 to 16 mg/l, makes it possibleto inhibit the sensitive gram-positive bacteria without interfering withthe detection of the ESBL bacteria.

The inhibition of the yeasts made it possible to improve the detectionof ESBL bacteria in polymicrobial samples and the specificity of theircoloration.

6. Conclusion

These results demonstrate that the medium according to the inventionmakes it possible to isolate and apparently identify ESBL-producingbacteria, differentiating them from high levelcephalosporinase-producing strains. The use of a biplate containing acephalosporine alone on one side and a cephalosporine/clavulanic acidcombination on the other, in a CPS ID 3 base, is particularlyadvantageous.

EXAMPLE 2

This second example is based on the phenotypic detection of ESBLs usingthe reduction of susceptibility to antibiotics and the sensitivity of HLCases to combinations with tobramycin or cloxacillin or dicloxacillin,and presents the use of a cephalosporine mentioned above (CTX, CAZ, CPD,CRO, ATM) in combination with a compound that inhibits cephalosporinases(cloxacillin, dicloxacillin and tobramycin). Such a medium makes itpossible to inhibit bacteria which have a “natural” cephalosporinase,most of those which have only a high level cephalosporinase (HL Case),while at the same time allowing growth of ESBL bacteria.

1. Choice of Strains

In the context of the manipulations carried out, for evaluating theactivity of antibiotics that are active on gram-negative bacilli,various species of enterobacteria (Escherichia coli, Enterobacteraerogenes, Klebsiella pneumoniae, Proteus mirabilis) and ofnonfermenting gram-active bacilli (Pseudomonas aeruginosa) capable ofproducing ESBLs, were used. In the assays, ESBL-positive strains,strains producing high level cephalosporinase (HL Case) and wild-typestrains are compared.

2. Preparation of the Medium

The medium used was a CPS ID3 medium (43541), also comprising:

-   -   ceftazidime at 2.5 mg/l and tobramycin at 2 mg/l (medium A) or    -   ceftriaxone at 2 mg/l and cloxacillin at 150 mg/l (medium B) or    -   cefpodoxime at 2 mg/l and dicloxacillin at a concentration of        between 500 and 1000 mg/l (medium C).

Osmosed water is added and the whole is homogenized and melted in awaterbath at 100° C.

The basic medium is dispensed into flasks, the number of whichcorresponds to the total number of media to be tested during theprocess. The flasks are then autoclaved for 15 min at 121° C. The mediaare brought back to and kept molten at 55±3° C. in a waterbath, in orderto sterilely add the thermolabile additives (sterilized beforehand byfiltration through 0.22 μm). The media are then poured into plates 35 mmin diameter and left on a flat surface so that they can set. The surfaceof the agars is then dried under a laminar flow hood for 30 min.

3. Inoculation of Media

This step is carried out as described in example 1.

4. Reading of Media

This step is carried out as described in example 1

5. Results

Medium A comprising ceftazidime and tobramycin made it possible toinhibit all the wild-type strains and all the HL Cases tested. Only theESBL-positive strains were detected on this medium.

Medium B comprising ceftriaxone and cloxacillin made it possible toinhibit all the HL Cases and all the wild-type strains except HL Caseand wild-type P aeruginosa, and grew only the majority of theESBL-positive strains.

Medium C comprising cefpodoxime and di-cloxacillin made it possible toinhibit all the wild-type strains and the majority of the HL Cases,without affecting the growth of ESBL-positive strains.

EXAMPLE 3

This third example is based on the phenotypic detection of enterococciresistant to glycopeptides, with specific distinction of Enterococcusfaecalis and E. faecium, using the reduction of susceptibility toantibiotics and the demonstration of an enzymatic activity:β-glucosidase, and of a metabolic activity: Methyl-α-glucosideacidification.

1. Choice of Strains

In the context of the manipulations carried out, for evaluating theactivity of the antibiotics active on enterococci, various species ofEnterococcus (Enterococccus faecalis, Enterococcus faecium, Enterococcuscasseliflavus, Enterococcus gallinarum,) were used. In the assays,strains resistant to glycopeptides (VRE) and wild-type strains arecompared.

2. Preparation of the Medium

The medium used was a Columbia medium (51026), also comprising:

-   -   5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside (X-Glu) at 100        mg/l,    -   methyl-α-D-glucoside at 9 g/l,    -   neutral red at 25 mg/l,    -   bilial salts at 5 g/l,    -   vancomycin at 4 mg/l,    -   amphotericin B at 2 mg/l.

Osmosed water is added and the whole is homogenized and melted in awaterbath at 100° C. The basic medium is dispensed into flasks, thenumber of which corresponds to the total number of media to be testedduring the process. The flasks are then autoclaved for 15 min at 121° C.The media are brought back to and kept molten at 55±3° C. in awaterbath, in order to sterilely add the thermolabile additives(sterilized beforehand by filtration through 0.22 μm). The media arethen poured into plates 90 mm in diameter and left on a flat surface sothat they can set. The surface of the agars is then dried under alaminar flow hood for 30 min.

3. Inoculation of Media

This step is carried out as described in example 1.

4. Reading of Media

This step is carried out as described in example 1.

5. Results:

On this medium, only the glycopeptide-resistant enterococcal strainsdevelop and form colonies.

The resistant E. faecalis and E. faecium strains form green colonies,whereas those of E. casseliflavus and of E. gallinarum formblue-to-violet colonies. This medium therefore makes it possible todifferentiate these two groups of enterococci and to provide a suitabletherapeutic response.

EXAMPLE 4

This fourth example is based on the phenotypic detection ofglycopeptide-resistant enterococci, with specific distinction ofEnterococcus faecalis and E. faecium, using the reduction ofsusceptibility to antibiotics and the demonstration of two enzymaticactivities: α-glucosidase and β-galactosidase or β-glucosidase.

1. Choice of Strains

In the context of the manipulations carried out, for evaluating theactivity of antibiotics active on enterococci, various species ofEnterococcus (Enterococccus faecalis, Enterococcus faecium, Enterococcuscasseliflavus, Enterococcus gallinarum,) were used. In the assays,strains resistant to glycopeptides (VRE) and wild-type strains arecompared.

2. Preparation of the Medium

The media used were a Columbia medium (51026), also comprising:

-   -   5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucopyranoside        (GreenA-α-Glu) at 150 mg/l,    -   6-chloro-3-indolyl-β-glucopyranoside (Rose-b-Glu [Pink-b-Glu])        at 200 mg/l,        or:    -   5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucopyranoside        (GreenA-α-Glu) at 150 mg/l,    -   alizarine-β-galactopyranoside at 50 mg/l

-   and vancomycin at 8 mg/l,

-   an amphotericin B at 4 mg/l,

-   and colistine at 2 mg/l,

-   and aztreonam at 32 mg/l.

Osmosed water is added and the whole is homogenized and melted in awaterbath at 100° C. The two basic media are dispensed into flasks. Theflasks are then autoclaved for 15 min at 121° C. The media are broughtback to and kept molten at 55±3° C. in a waterbath, in order tosterilely add the thermolabile additives (sterilized beforehand byfiltration through 0.22 μm). The media are then poured into plates 90 mmin diameter and left on a flat surface so that they can set. The surfaceof the agars is then dried under a laminar flow hood for 30 min.

3. Inoculation of Media

This step is carried out as described in example 1.

4. Reading of Media

This step is carried out as described in example 1.

5. Results

On the medium containing a substrate for α-glucosidase and forβ-glucosidase, the resistant E. faecium strains form violet colonies,whereas the resistant E. faecalis strains form pink colonies. The E.casseliflavus and E. gallinarum strains (natural resistances) areinhibited due to the concentration of vancomycin.

This medium therefore makes it possible to differentiate these twogroups of enterococci and to provide a suitable therapeutic response andalso a follow-up of the local epidemiology.

On the medium containing a substrate for α-glucosidase and forβ-galactosidase, the resistant E. faecium strains form violet colonies,whereas the resistant E. faecalis strains form green colonies. The E.casseliflavus and E. gallinarum strains (natural resistances) areinhibited due to the concentration of vancomycin.

This medium therefore makes it possible to differentiate these twogroups of enterococci and to provide a suitable therapeutic response anda follow-up of the local epidemiology.

What is claimed is:
 1. A method for distinguishing among at least threegroups of microorganisms that may be present in a biological sample, theat least three groups comprising: a first group of microorganisms,belonging to a first taxon of bacteria that is vancomycin resistant; asecond group of microorganisms, belonging to a second taxon ofvancomycin resistant bacteria that is different than said first taxon;and a third group of microorganisms that is not resistant or has anatural resistance to vancomycin, the method comprising: inoculating aculture medium with the biological sample, the culture mediumcomprising: a first substrate for detecting a first enzymatic activityof said first group of microorganisms; a marker for differentiating thefirst group of microorganisms and the second group of microorganisms,said marker being a substrate for detecting an enzymatic activity ofsaid second group of microorganisms; and an antimicrobial that is activeon the third group of microorganisms, and distinguishing among anymembers of the three groups of microorganisms that are present on theculture medium to determine of which group of microorganisms they aremembers on the basis of their interactions with the first substrate, themarker, and the antimicrobial.
 2. The method of claim 1, wherein: thefirst group of microorganisms is enterococcal bacteria that have anacquired resistance to vancomycin; the second group of microorganisms isenterococcal bacteria that have a natural resistance to vancomycin; andthe third group of microorganisms is enterococcal bacteria that is notresistant to vancomycin.
 3. The method of claim 1, wherein: the firstgroup of vancomycin-resistant microorganisms comprises Enterococcusfaecalis and Enterococcus faecium; the second group ofvancomycin-resistant microorganisms comprises Enterococcus casseliflavusand Enterococcus gallinarum; and the third group of microorganisms isnot resistant to vancomycin.
 4. The method of claim 1, wherein: thefirst group of vancomycin-resistant microorganisms comprisesEnterococcus faecium; the second group of vancomycin-resistantmicroorganisms comprises Enterococcus faecalis; and the third group ofmicroorganisms is enterococcal bacteria that is not resistant tovancomycin or that have a natural resistance to vancomycin.
 5. Themethod of claim 1, wherein: the first group of vancomycin-resistantmicroorganisms comprises Enterococcus faecalis and Enterococcus faecium;the second group of vancomycin-resistant microorganisms comprisesStaphylococcus aureus that is intermediary resistant or resistant tovancomycin; and the third group of microorganisms is not resistant tovancomycin.
 6. The method of claim 1, wherein: the first substrate is anenzymatic substrate for detecting alpha-glucosidase activity; the markeris a second substrate for detecting a second activity different thanalpha-glucosidase activity; and the antimicrobial is vancomycin.
 7. Themethod of claim 6, wherein the second substrate detects beta-glucosidaseor beta-galactosidase activity.
 8. The method of claim 6, wherein: thefirst substrate is 5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucoside or5-bromo-4-chloro-3-indolyl-α-D-glucoside, and the second substrate is6-chloro-3-indolyl-β-D-glucoside, alizarine-β-D-galactoside,5-bromo-6-chloro-3-indolyl-β-D-glucoside,5-bromo-6-chloro-3-indolyl-β-D-galactoside or6-chloro-3-indolyl-β-D-galactoside.
 9. The method of claim 6, wherein:the first substrate is 5-bromo-4-chloro-3-indolyl-N-methyl-α-D-glucosideor 5-bromo-4-chloro-3-indolyl-α-D-glucoside, and the second substrate is6-chloro-3-indolyl-β-D-glucoside or5-bromo-6-chloro-3-indolyl-β-D-glucoside.
 10. The method of claim 1,wherein: the first group of vancomycin-resistant microorganismscomprises Enterococcus faecalis and Enterococcus faecium; the secondgroup of vancomycin-resistant microorganisms comprises Enterococcuscasseliflavus and Enterococcus gallinarum; the third group ofmicroorganisms is not resistant to vancomycin; the first substrate is anenzymatic substrate for detecting alpha-glucosidase activity; the markeris a second substrate for detecting a second activity different thanalpha-glucosidase activity; and the antimicrobial is vancomycin.
 11. Themethod of claim 1, wherein: the first group of vancomycin-resistantmicroorganisms comprises Enterococcus faecium; the second group ofvancomycin-resistant microorganisms comprises Enterococcus faecalis; thethird group of microorganisms is enterococcal bacteria that is notresistant to vancomycin or that have a natural resistance to vancomycin;the first substrate is an enzymatic substrate for detectingalpha-glucosidase activity; the marker is a second substrate fordetecting a second activity different than alpha-glucosidase activity;and the antimicrobial is vancomycin.
 12. The method of claim 1, wherein:the first group of vancomycin-resistant microorganisms comprisesEnterococcus faecalis and Enterococcus faecium; the second group ofvancomycin-resistant microorganisms comprises Staphylococcus aureus thatis intermediary resistant or resistant to vancomycin; the third group ofmicroorganisms are not resistant to vancomycin; the first substrate isan enzymatic substrate for detecting alpha-glucosidase activity; themarker is a second substrate for detecting a second activity differentthan alpha-glucosidase activity; and the antimicrobial is vancomycin.13. The method of claim 1, wherein: the first group ofvancomycin-resistant microorganisms comprises Enterococcus faecalis andEnterococcus faecium; the second group of vancomycin-resistantmicroorganisms comprises Enterococcus casseliflavus and Enterococcusgallinarum; the third group of microorganisms is not resistant tovancomycin; the first substrate is an enzymatic substrate for detectingalpha-glucosidase activity; the marker is a second substrate fordetecting beta-glucosidase or beta-galactosidase activity; and theantimicrobial is vancomycin.
 14. The method of claim 1, wherein: thefirst group of vancomycin-resistant microorganisms comprisesEnterococcus faecium; the second group of vancomycin-resistantmicroorganisms comprises Enterococcus faecalis; the third group ofmicroorganisms is enterococcal bacteria that is not resistant tovancomycin or that have a natural resistance to vancomycin; the firstsubstrate is 5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or5-bromo-4-chloro-3-indolyl-a-D-glucoside; the marker is6-chloro-3-indolyl-β-D-glucoside, alizarine-β-D-galactoside,5-bromo-6-chloro-3-indolyl-β-D-glucoside,5-bromo-6-chloro-3-indolyl-β-D-galactoside or6-chloro-3-indolyl-β-D-galactoside; and the antimicrobial is vancomycin.15. The method of claim 1, wherein: the first group ofvancomycin-resistant microorganisms comprises Enterococcus faecalis andEnterococcus faecium; the second group of vancomycin-resistantmicroorganisms comprises Staphylococcus aureus that is intermediaryresistant or resistant to vancomycin; the third group of microorganismsis not resistant to vancomycin; the first substrate is5-bromo-4-chloro-3-indolyl-N-methyl-a-D-glucoside or5-bromo-4-chloro-3-indolyl-a-D-glucoside; the marker is6-chloro-3-indolyl-β-D-glucoside or5-bromo-6-chloro-3-indolyl-β-D-glucoside; and the antimicrobial isvancomycin.
 16. A method of detecting and distinguishing between anyextended-spectrum β-lactamase-producing E. coli bacteria (ESBL E. colibacteria) and any extended-spectrum β-lactamase-producing KESC bacteria(ESBL KESC bacteria) that are present on a culture medium, the methodcomprising: inoculating a culture medium with a biological sample,wherein the culture medium comprises: a first substrate for detectingbeta-galactosidase activity; a second substrate for detectingbeta-glucuronidase activity; a third substrate for detecting desaminaseactivity; and a combination of antimicrobials throughout the entireculture medium, the combination comprising a cephalosporinase inhibitorand a third-generation cephalosporin; and simultaneously detecting anddistinguishing between any ESBL E. coli bacteria and ESBL KESC bacteriathat are present on the culture medium.
 17. A method of detecting anddistinguishing between any extended-spectrum β-lactamase-producing E.coli bacteria (ESBL E. coli bacteria) and any extended-spectrumβ-lactamase-producing KESC bacteria (ESBL KESC bacteria) that arepresent on a culture medium, the method comprising: inoculating aculture medium with a biological sample, wherein the culture mediumcomprises: a first substrate for detecting beta-galactosidase activity;a second substrate for detecting beta-glucosidase activity; and acombination of antimicrobials throughout the entire culture medium, thecombination comprising a cephalosporinase inhibitor and athird-generation cephalosporin.